Cloning and sequencing of allergens of dermatophagoides house dust mite

ABSTRACT

Isolated DNA encoding allergens of Dermatophagoides (house dust mites) particularly of the species  Dermatophagoides farinae  and  Dermatophagoides pteronyssinus , which are protein allergens or peptides which include at least one epitope of the protein allergen. In particular, DNA encoding two major  D. farinae  allergens,  Der f  I and  Der f  II and DNA encoding a  D. pteronyssinus  allergen,  Der p  I. In addition, the proteins or peptides encoded by the isolated DNA, their use as diagnostic and therapeutic reagents and methods of diagnosing and treating sensitivity to house dust mite allergens.

RELATED APPLICATION

This application is a continuation of application Ser. No. 08/175,071,filed Dec. 29, 1993, now abandoned, which is a divisional of applicationSer. No. 08/107,332, filed Aug. 16, 1993, now abandoned which is acontinuation of application Ser. No. 07/580,655, filed on Sep. 11, 1990,now abandoned, which is a Continuation-In-Part of U.S. Ser. No. 458,642entitled “Cloning of Mite Allergens”, by Wayne Robert Thomas, GeoffreyAlexander, Stewart Keven James Turner and Richard John Simpson (filedFeb. 13, 1990), now abandoned. The teachings of application U.S. Ser.No. 458,642 are incorporated herein by reference.

FUNDING

Work described herein was funded by grants from the Princess MargaretChildren's Medical Research Foundation, the Australian Health andMedical Research Council and the Asthma Foundation of Australia.

BACKGROUND

Recent reports have documented the importance of responses to the GroupI and Group II allergens in house dust mite allergy. For example, it hasbeen documented that over 60% of patients have at least 50% of theiranti-mite antibodies directed towards these proteins (Lind, P. et al.,Allergy, 39:259-274 (1984); van der Zee, J. S. et al., J. Allergy Clin.Immunol., 81:884-896 (1988)). It is possible that children show agreater degree of reactivity (Thompson, P. J. et al, Immunology,64:311-314 (1988)). Allergy to mites of the genus Dermatophagoides (D.)is associated with conditions such as asthma, rhinitis and ectopicdermatitis. Two species, D. pteronyssinus and D. farinae, predominateand, as a result, considerable effort has been expended in trying toidentify the allergens produced by these two species. D. pteronyssinusmites are the most common Dermatophagoides species in house dust inWestern Europe and Australia. The species D. farinae predominates inother countries, such as, North America and Japan (Wharton, G. W., J.Medical Entom, 12:577-621 (1976)). It has long been recognized thatallergy to mites of this genus is associated with diseases such asasthma, rhinitis and atopic dermatitis. It is still not clear whatallergens produced by these mites are responsible for the allergicresponse and associated conditions.

SUMMARY OF THE INVENTION

The present invention relates to isolated DNA which encodes a proteinallergen of Dermatophagoides (D.) house dust mite) or a peptide whichincludes at least one epitope of a protein allergen of a house dust miteof the genus Dermatophagoides. It particularly relates to DNA encodingmajor allergens of the species D. farinae, designated Der f I and Der fII, or portions of these major allergens (i.e., peptides which includeat least one epitope of Der f I or of Der f II). It also particularlyrelates to DNA encoding major allergens of D. pteronyssinus, designatedDer p I and Der p II, or portions of these major allergens (i.e.,peptides which include at least one epitope of Der p I or of Der p II.

The present invention further relates to proteins and peptides encodedby the isolated Dermatophagoides (e.g., D. farinae, D. pteronyssinus)DNA. Peptides of the present invention include at least one epitope of aD. farinae allergen (e.g., at least one epitope of Der f I or of Der fII) or at least one epitope of a D. pteronyssinus allergen (e.g., atleast one epitope of Der p I or of Der p II). It also relates toantibodies specific for D. farinae proteins or peptides and toantibodies specific for D. pteronyssinus proteins or peptides.

Dermatophagoides DNA, proteins and peptides of the present invention areuseful for diagnostic and therapeutic purposes. For example, isolated D.farinae protein or peptide can be used to detect sensitivity in anindividual to house dust mites and can be used to treat sensitivity(reduce sensitivity or desensitize) in an individual, to whomtherapeutically effective quantities of the D farinae protein or peptideis administered. For example, isolated D. farinae protein allergen, suchas Der f I or Der f II can be administered periodically, using standardtechniques, to an individual in order to desensitize the individual.Alternatively, a peptide which includes at least one epitope of Der f Ior of Der f II can be administered for this purpose. Isolated D.pteronyssinus protein allergen, such as Der p I or Der p II, can beadministered as described for Der f I or Der f II. Similarly a peptidewhich includes at least one Der p I epitope or at least one Der p IIepitope can be administered for this purpose. A combination of theseproteins or peptides (e.g., Der f I and Der f II; Der p I and Der p II;or a mixture of both Der f and Der p proteins) can also be administered.The use of such isolated proteins or peptides provides a means ofdesensitizing individuals to important house dust mite allergens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of the cDNA insert of clone λgt11 f 1,including a schematic representation of the strategy of DNA sequencing.Arrows indicate directions in which sequences were read.

FIG. 2 is the nucleotide sequence and the predicted amino acid sequenceof cDNA λgt11 f 1. Numbers above are nucleotide positions; numbers tothe left are amino acid positions. Positive amino acid residue numberscorrespond to the sequence of the mature excreted Der f I beginning withthreonine. Negative sequence numbers refer to the signal peptide and theproenzyme regions of Der f I. The arrows indicate the beginning of theproenzyme sequence and the mature Der f I, respectively. The underlinedresidues −81 to −78 make up the proposed cleavage site for the proenzymeformation, while the underlined residues 53-55 represent a potentialN-glycosylation site. The termination TGA codon and the adjacentpolyadenylation signal are also underlined. Amino acid residues 1-28correspond to a known tryptic peptide sequence determined byconventional amino acid sequencing analysis.

FIG. 3 is a composite alignment of the amino acid sequences of themature Der p I and Der f I proteins. The numbering above the sequencerefers to Der p I. The asterisk denotes the gap that was introduced formaximal alignment. The symbol (.) is used to indicate that the aminoacid residue of Der f I at that position is identical to thecorresponding amino acid residue of Der p I. The arrows indicate thoseresidues making up the active site of Der p I and Der f I.

FIG. 4 is a comparison of the amino acid sequence the pre- andpro-peptide regions of Der f I with those of rat cathepsin H, ratcathepsin L, papain, aleurain, CP1, CP2, rat cathepsin B, CTLA-2, MCP,Der p I and actinidin. Gaps, denoted by dashes, were added for maximalalignment. Double asterisks denote conserved amino acid residues whichare shared by greater than 80% of the proenzymes; single asterisks showresidues which are conserved in greater than 55% of the sequences. Thesymbol (.) is used to denote semiconserved equivalent amino acids whichare shared by greater than 90% of the proenzyme regions.

FIG. 5 is a hydrophilicity plot of the Der p I mature protein and ahydrophilicity plot of the Der f I mature protein produced using theHopp-Woods algorithm computed with the Mac Vector Sequence AnalysisSoftware (IBI, New Haven) using a 6 residue window. Positive valuesindicate relative hydrophilicity and negative values indicating relativehydrophobicity.

FIG. 6 is the nucleotide sequence and the predicted amino acid sequenceof Der f II cDNA. Numbers to the right are nucleotide positions andnumbers above are amino acid residues. The stop (TAA) signal isunderlined. The first 8 nucleotides are from the oligonucleotide primerused to generate the cDNA, based on the Der p II sequence.

FIG. 7 is the nucleotide sequence and predicted amino acid sequence ofcDNA λgt11 p1(13T). Numbers to the right are nucleotides and numbersabove are amino acid positions. Positive amino acids correspond to thesequence of mature excreted Der p I beginning with threonine.

FIG. 8 is a restriction map of Der f II cDNA, which was generated bycomputer from the sequence data. A map of Der p II similarly generatedis shown for comparison. There are few common restriction enzyme sitesconserved. Sites marked with an asterisk were introduced by cloningprocedures.

FIG. 9 hows the alignment of Der f II and Der p II cDNA sequences.Numbers to the right are nucleotide position and numbers above and aminoacid residues. The top line gives the Der p II nucleotide sequence andthe second the Der p II amino acid residues. The next two lines showdifferences of Der f II to these sequences.

FIG. 10 is a hydrophilicity plot of Der f II and Der p II using theHopp-Woods algorithm computed with the Mac Vector Sequence AnalysisSoftware (IBI, New Haven) using a 6-residue window.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nucleotide sequence coding for anallergen from the house dust mite Dermatophagoides and to the encodedDermatophagoides protein or peptide which includes at least one epitopeof the Dermatophagoides allergen. It particularly relates to anucleotide sequence capable of expression in an appropriate host of amajor allergen of D. farinae, such as Der f I or Der f II, or of apeptide which includes at least one epitope of Der f I or of Der f II.It also particularly relates to a nucleotide sequence capable ofexpression in an appropriate host of a major allergen of D.pteronyssinus, such as Der p I or Der p II, or of a peptide whichincludes at least one epitope of Der p I or of Der p II. TheDermatophagoides nucleotide sequence is useful as a probe foridentifying additional nucleotide sequences which hybridize to it andencode other mite allergens, particularly D. farinae or D. pteronyssinusallergens. Further, the present invention relates to nucleotidesequences which hybridize to a D. farinae protein-encoding nucleotidesequence or a D. pteronyssinus protein-encoding nucleotide sequence butwhich encode a protein from another species or type of house dust mite,such as D. microceras (e.g., Der m I and Der m II).

The encoded Dermatophagoides mite allergen or peptide which includes atleast one Dermatophagoides (Der f I or Der f II; Der p I or Der p II)epitope can be used for diagnostic purposes (e.g., as an antigen) andfor therapeutic purposes (e.g., to desensitize an individual).Alternatively, the encoded house dust mite allergen can be a protein orpeptide, such as a D. microceras protein or peptide, which displays theantigenicity of or is cross-reactive with a Der f or a Der p allergen;generally, these have a high degree of amino acid homology.

Accordingly, the present invention also relates to compositions whichinclude a Dermatophagoides allergen (e.g., Der f I or allergen, Der f IIallergen; Der p I or Der p II allergen or other D. allergencross-reactive therewith) or a peptide which includes at least oneepitope of a Dermatophagoides allergen (Der f I, Der f II, Der p I, Derp II or other D. allergen cross-reactive therewith) individually or incombination, and which can be used for therapeutic applications (e.g.,desensitization). As is described below, DNA coding for major allergensfrom house dust mites have been isolated and sequenced. In particular,and as is described in greater detail in the Examples, two cDNA clones,coding, respectively, for Der f I allergen and Der f II allergen, havebeen isolated and sequenced. The nucleotide sequences of Der p I and Derp II have also been determined. The nucleotide sequence of each of theseclones has been compared with that of the homologous allergen from therelated mite D. pteronyssinus (Der p I and Der p II, respectively), ashas the predicted amino acid sequence of each.

The following is a description of isolation and sequencing of the twocDNA clones coding for Der f allergens and their comparison with thecorresponding D. pteonyssinus allergen and a description of use of thenucleotide sequences and encoded products in a diagnostic or atherapeutic context.

Isolation and Sequence Analysis of Der f I

A cDNA clone coding for Der f I, a major allergen from the house dustmite D. farinae, has been isolated and sequenced. A restriction map ofthe cDNA insert of the clone is represented in FIG. 1, as is thestrategy of DNA sequencing. This Der f I cDNA clone contains a 1.1-kbcDNA insert encoding a typical signal peptide, a proenzyme region andthe mature Der f I protein. The product is 321 amino acid residues: aputative 18 residue signal peptide, an 80 residue proenzyme(pro-peptide) region, and a 223 residue mature enzyme region. Thederived molecular weight is 25,191. The nucleotide sequence and thepredicted amino acid sequence of the Der f I cDNA are represented inFIG. 2. The deduced amino acid sequence shows significant homology toother cysteine proteases in the pro-region, as well as in the matureprotein. Sequence alignment of the mature Der f I protein with thehomologous allergen Der p I from the related mite D. pteronyssinus (FIG.3) revealed a high degree of homology (81%) between the two proteins, aspredicted by previous sequencing at the protein level. In particular,the residues comprising the active site of these enzymes were conservedand a potential N-glycosylation site was present at equivalent positionsin both mite allergens.

Conserved cysteine residue pairs (31, 71) and (65, 103), where thenumbering refers to Der p I, are apparently involved in disulphide bondformation on the basis of the assumed similarity of the threedimensional structure of Der p I and Der f I to that of papain andactinidin, which also have an additional disulphide bridge. The fifthand final cysteine residue for which there is a homologous cysteineresidue in papain and actinidin is the active site cysteine (residue 35in Der f I). It is not unlikely that the two extra cysteine residuespresent in Der p I and Der f I may be involved in forming a thirddisulphide bridge.

The potential N-glycosylation site in Der p I is also present at theequivalent position in Der f I, with conservation of the crucial firstand last residues of the tripeptide site. The degree of glycosylation ofDer f I and Der p I has yet to be determined. Carbohydrates, includingmannose, galactose, N-acetylglucosamine and N-acetylgalactosamine, havebeen reported in purified preparations of these mite allergens (Chapman,M. D., J. Immunol., 125:587-592 (1980); Wolden, S. et al., Int. Arch.Allergy Appl. Immunol., 68:144-151 (1982)).

Given the degree of homology over the first thirty N-terminal amino acidresidues between mature Der p I and Der m I (70%) and mature Der f I andDer m I (97%) with the Der m I residues determined by conventional aminoacid sequencing (Platts-Mills TAE et al., In: Mite Allergy, a World-WideProblem, 27-29 (1988); Lind, P. and N. Horn, In: Mite Allergy, aWorld-Wide Problem, 30-34 (1988)), it is probable that the full matureDer m I sequence will confirm an overall 70-80% homology between theGroup I mite allergens. Der m I is an allergen from D. microceras. Highhomology between the proenzyme moieties of Der p I and Der f I (91%)over the residues −23 to −1 and the structural analysis of Der f Isuggests that the Group I allergens are likely to have N-terminalextension peptides of the mature protein of homologous structure and, atleast for the pro-peptide, composition.

Studies on the fine structure of the design of signal sequences haveidentified three structurally dissimilar regions so far: a positivelycharged N-terminal (n) region, a central hydrophobic (h) region and amore polar C-terminal (c) region that seems to define the cleavage site(Von Heijne, G., EMBO J., 3:2315-2323 (1984); Eur. J. Biochem.,133:17-21 (1983); J. Mol. Biol., 184:99-105 (1985)). Analysis of thesignal peptide of Der f I revealed that it, too, contained these regions(FIG. 4). The n-region is extremely variable in length and composition,but its net charge does not vary appreciably with the overall length,and has a mean value of about +1.7. The n-region of the Der f I signalpeptide, with a length of two residues, has a net charge of +2contributed by the initiator methionine (which is unformylated and hencepositively charged in eukaryotes) and the adjacent lysine (Lys) residue.The h-region of Der f I is enriched with hydrophobic residues, thecharacteristic feature of this region, with only one hydrophilic residueserine (Ser) present which can be tolerated. The overall amino acidcomposition of the Der f I c-region is more polar than that of theh-region as is found in signal sequences with the h/c boundary locatedbetween residues −6 and −5, which is its mean position in eukaryotes.Thus, the Der f I pre-peptide sequence appears to fulfill therequirements to which a functional signal sequence must conform.

While the signal sequence of Der f I and other cysteine proteases sharestructural homology, all being composed of the n, h and c-regions, theyare highly variable with respect to overall length and amino acidsequence, as is clear in FIG. 4. However, significant sequence homologyhas been shown between the pro-regions of cysteine protease precursors(Ishidoh, K. et al., FEBS Letters, 226:33-37 (1987)). Alignment of theproenzyme regions of Der f I and a number of other cysteine proteases(FIG. 4) indicated that these proregions share a number of veryconserved residues as well as semi-conserved residues which were presentin over half of the sequences. This homology was increased ifconservative amino acids such as valine (Val), isoleucine (Ile) andleucine (Leu) (small hydrophobic residues) or arginine (Arg) and Lys(positively charged residues) were regarded as identical. The Der f Iproregion possessed six out of the seven highly conserved amino acidsand all the residues at sites of conservative changes. The homology atless conserved sites was lower. Homology in the pro-peptide, inparticular the highly conserved residues, may be important whenconsidering the function of the pro-peptide in the processing of theseenzymes, since it indicates that these sequences probably havestructural and functional similarities.

Highly cross-reactive B cell epitopes on Der f I and Der p I have beendemonstrated using antibodies present in mouse, rabbit and human sera(Heymann, P. W. et al., J. Immunol, 137:2841-2847 (1986); Platts-Mills,TAE et al., J. Allergy Clin. Immunol., 78:398-407 (1986)). However,species-specific epitopes have also been defined in these systems.Murine monoclonal antibodies bound pre-dominantly to species-specificdeterminants (Platts-Mills TAE et al., J. Immunol,. 139:1479-1484(1987). Some 40% of rabbit anti-Der p I reactivity was accounted for byepitopes unique to Der p I (Platts-Mills, TAE et al., J. Allergy Clin.Immunol., 78:398-407 (1986)), and some species-specific binding ofantibodies from allergic humans was observed, although the majority bindto cross-reactive epitopes (Platts-Mills TAE et al., J. Immunol.,139:1479-1484 (1987).

The recombinant DNA strategy of gene fragmentation and expression wasused (Greene, W. K. et al., Immunol. (1990)) to define five antigenicregions of recombinant Der p I which contained B cell epitopesrecognized by a rabbit anti-Der p I antiserum. Using the technique ofimmunoabsorption, three of these putative epitopes were shown to beshared with Der f I (located on regions containing amino acid residues34-47, 60-72 and 166-194) while two appeared to be specific for Der p I(regions 82-99 and 112-140). Differences in the reactivity of thesepeptides to rabbit anti-D. farinae supported the above division intocross-reactive and species-specific epitopes. The sequence differencesshown between the Der p I and the Der f I proteins are primarily locatedin the N and C terminal regions, as well as in an extended surface loop(residues 85-136) linking the two domains of the enzyme that includeshelix D (residues 127-136), as predicted from the secondary and tertiarystructures of papain and actinidin (Baker, E. N. and J. Drenth, In:Biological Macromolecules and Assemblies, Vol. 3, pp. 314-368, JohnWiley and Sons, NY. (1987)). The surface location of these residues issupported by the hydrophilicity plots of Der p I and Der f I in FIG. 5,which illustrate the predominantly hydrophilic nature of this regionthat predicts surface exposure. This region also contains the twospecies-specific B cell epitopes recognized by the rabbit anti-Der p Iserum (see above). Analysis of the sequences in the regions containingthe cross-reactive epitopes showed that two of the cross-reactiveepitopes (located in regions 34-47 and 60-72) are completely conservedbetween Der p I and Der f I, while the majority of residues in a thirdcross-reactive epitope-containing region (residues region 166-194) wereconserved.

Expression of results in production of pre-pro-Der f I protein in E.coli a recombinant protein of greater solubility, stability andantigenicity than that of recombinant Der p I. Protein encoded by Der fI cDNA has been expressed using a pGEX vector and has been shown byradioimmune assay to react with rabbit anti-D. farinae antibodies. Theavailability of high yields of soluble Der f I allergen and antigenicderivatives will facilitate the development of diagnostic andtherapeutic agents and the mapping of B and T cell antigenicdeterminants.

With the availability of the complete amino acid sequence of recombinantDer f I, mapping of the epitopes recognized by both the B and T cellcompartments of the immune system can be carried out. The use oftechniques such as the screening of overlapping synthetic peptides, theuse of monoclonal antibodies and gene fragmentation and expressionshould enable the identification of both the continuous andtopographical epitopes of Der f I. It will be particularly useful todetermine whether allergenic (IgE-binding) determinants have commonfeatures and are intrinsically different from antigenic (IgG-binding)determinants and whether T cells recognize unique epitopes differentfrom those recognized by B cells. Studies to identify the Der f Iepitopes reactive with mite allergic human IgE antibodies and thedivision of these into determinants cross-reactive with Der p I anddeterminants unique to Der f I can also be carried out. B cell (and Tcell) epitopes specific for either species can be used to provide usefuldiagnostic reagents for determining reactivity to the different mitespecies, while cross-reacting epitopes are candidates for a commonimmuno-therapeutic agent.

As described in co-pending Application U.S. Ser. No. 458,643,incorporated herein by reference, the molecular cloning of miteallergens resulted in the isolation of a cDNA clone coding for Der p Iwhich contained a 0.8-kb cDNA insert. Sequence analysis revealed thatthe 222 amino acid residue mature recombinant Der p I protein showedsignificant homology with a group of cysteine proteases, includingactinidin, papain, cathepsin H and cathepsin B.

Isolation and Sequence Analysis of Der f II

A cDNA clone coding for Der f II, a major allergen from the house dustmite D. farinae, has been isolated and sequenced, as described inExample 2. The nucleotide sequence and the predicted amino acid sequenceof the Der f II cDNA are represented in FIG. 6. A restriction map of thecDNA insert of a clone coding for Der f II is represented in FIG. 8.

FIG. 9 shows the alignment of Der f II and Der p II cDNA sequences.

The homology of the sequence of Der f II with Derby II (88%) is higherthan the 81% homology found with Der p I and Der f I, which issignificantly different (p<0.05) using the chi² distribution. The reasonfor this may simply be that the Group I allergens are larger and eachresidue may be less critical for the structure and function of themolecule. It is known; for example, that assuming they adopt a similarconformation to other cysteine proteases, many of the amino aciddifferences in Der p I and Der f I lie in residues linking the twodomain structures of the molecules. The 6 cysteine molecules areconserved between the group II allergens, suggesting a similardisulphide bonding, although this may be expected, given the highoverall homology. Another indication of the conservation of theseproteins is that 34/55 of the nucleotide changes of the coding sequenceare in the third base of a codon, which usually does not change theamino acid. Residues that may be of importance in the function of themolecule are Ser 57 where all three bases are changed but the amino acidis conserved. A similar phenomenon exists at residue 88, where acomplete codon change has conserved a small aliphatic residue. Again,like Der p II, the Der f II cDNA clone does not have a poly A tail,although the 3′ non-coding region is rich in adenosine and has twopossible polyadenylation signals ATAA. The nucleotides encoding thefirst four residues are from the PCR primer which was designed from theknown homology of Der p II and Der f II from N-terminal amino acidsequencing. A primer based on the C-terminal sequence can now be used todetermine these bases, as well as the signal sequence.

Uses of the Subject Allergenic Proteins/Peptides and DNA Encoding Same

The materials resulting from the work described herein, as well ascompositions containing these materials, can be used in methods ofdiagnosing, treating and preventing allergic responses to miteallergens, particularly to mites of the genus Dermatophagoides, such asD. farinae and other species (e.g., D. pteronyssinus and D. microceras).In addition, the cDNA (or the mRNA from which it was transcribed) can beused to identify other similar sequences. This can be carried out, forexample, under conditions of low stringency and those sequences havingsufficient homology (generally greater than 40%) can be selected forfurther assessment using the method described herein. Alternatively,high stringency conditions can be used. In this manner, DNA of thepresent invention can be used to identify sequences coding for miteallergens having amino acid sequences similar to that of Der f I, Der fII, Der p I or Der p II. Thus, the present invention includes not onlyDer f and Der p II, but other mite allergens as well (e.g., other miteallergens encoded by DNA which hybridizes to DNA of the presentinvention).

Proteins or peptides encoded by the cDNA of the present invention can beused, for example, as “purified” allergens. Such purified allergens areuseful in the standardization of allergen extracts or preparations whichcan be used as reagents for the diagnosis and treatment of allergy tohouse dust mites. Through use of the peptides of the present invention,allergen preparations of consistent, well-defined composition andbiological activity can be made and administered for therapeuticpurposes (e.g., to modify the allergic response of a house dustmite-sensitive individual). Der f I or Der f II peptides or proteins (ormodified versions thereof, such as are described below) may, forexample, modify B-cell response to Der f I or Der f II, T-cell responseto Der f I and Der f II, or both responses. Similarly, Der p I or Der pII proteins or peptides may be used to modify B-cell and/or T-cellresponse to Der p I or Der p II. Purified allergens can also be used tostudy the mechanism of immunotherapy of allergy to house dust mites,particularly to Der f I, Der f II, Der p I and Der p II, and to designmodified derivatives or analogues which are more useful in immunotherapythan are the unmodified (“naturally-occurring”) peptides.

In those instances in which there are epitopes which are cross-reactive,such as the three epitopes described herein which are shared by Der f Iand Der p I, the area(s) of the molecule which contain thecross-reactive epitopes can be used as common immunotherapeutic peptidesto be administered in treating allergy to the two (or more) mite specieswhich share the epitope. For example, the cross-reactive epitopes couldbe used to induce IgG blocking antibody against both allergens (e.g.,Der f I and Der p I allergen). A peptide containing a univalent antibodyepitope can be used, rather than the entire molecule, and may proveadvantagious because the univalent antibody epitope cannot crosslinkmast cells and cause adverse reactions during desensitizing treatments.It is also possible to attach a B cell epitope to a carrier molecule todirect T cell control of allergic responses.

Alternatively, it nay be desirable or necessary to have peptides whichare specific to a selected Dermatophagoides allergen. As describedherein, two epitopes which are apparently Der p I-specific have beenidentified. A similar approach can be used to identify otherspecies-specific epitopes (e.g., Der p I or II, Der f I or II. Thepresence in an individual of antibodies to the species-specific epitopescan be used as a quick serological test to determine which mite speciesis causing the allergic response. This would make it possible tospecifically target therapy provided to an individual to the causativespecies and, thus, enhance the therapeutic effect.

Work by others has shown that high doses of allergens generally producethe best results (i.e., best symptom relief). However, many people areunable to tolerate large doses of allergens because of allergicreactions to the allergens. Modification of naturally-occurringallergens can be designed in such a manner that modified peptides ormodified allergens which have the same or enhanced therapeuticproperties as the corresponding naturally-occurring allergen but havereduced side effects (especially, anaphylactic reactions) can beproduced. These can be, for example, a peptide of the present invention(e.g., one having all or a portion of the amino acid sequence of Der f Ior Der f II, Der p I or Der p II). Alternatively, a combination ofpeptides can be administered. A modified peptide or peptide analogue(e.g., a peptide in which the amino acid sequence has been altered tomodify immunogenicity and/or reduce allergenicity or to which acomponent has been added for the same purpose) can be used fordesensitization therapy.

Administration of the peptides of the present invention to an individualto be desensitized can be carried out using known techniques. A peptideor combination of different peptides can be administered to anindividual in a composition which includes, for example, an appropriatebuffer, a carrier and/or an adjuvant. Such compositions will generallybe administered by injection, inhalation, transdermal application orrectal administration. Using the information now available, it ispossible to design a Der f I or Der f II peptide which, whenadministered to a sensitive individual in sufficient quantities, willmodify the individual's allergic response to a Der f I and/or Der f II.This can be done, for example, by examining the structures of Der f I orDer f II, producing peptides to be examined for their ability toinfluence B-cell and/or T-cell responses in house dust mite-sensitiveindividuals and selecting appropriate epitopes recognized by the cells.Synthetic amino acid sequences which mimic those of the epitopes andwhich are capable of down regulating allergic response to Der f I or Derf II allergen can be made. Proteins, peptides or antibodies of thepresent invention can also be used, in known methods, for detecting anddiagnosing allergic response to Der f I or Der f II. For example, thiscan be done by combining blood obtained from an individual to beassessed for sensitivity to one of these allergens with an isolatedallergenic peptide of house dust mite, under conditions appropriate forbinding of or stimulating components (e.g., antibodies, T cells, Bcells) in the blood with the peptide and determining the extent to whichsuch binding occurs. The Der p I and Der p II proteins or peptides canbe used in a similar manner for desensitization and diagnosis ofsensitivity. Der f and Der p proteins or peptides can be administeredtogether to treat an individual sensitive to both allergen types.

It is now also possible to design an agent or a drug capable of blockingor inhibiting the ability of Der f I or Der f II to induce an allergicreaction in house dust mite-sensitive individuals. Such agents could bedesigned, for example, in such a manner that they would bind to relevantanti-Der f I or anti-Der f II IgEs, thus preventing IgE-allergen bindingand subsequent mast cell degranulation. Alternatively, such agents couldbind to cellular components of the immune system, resulting insuppression or desensitization of the allergic response to theseallergens. A non-restrictive example of this is the use of appropriateB- and T-cell epitope peptides, or modifications thereof, based on thecDNA/protein structures of the present invention to suppress theallergic response to Der f I or Der f II allergens. This can be carriedout by defining the structures of B- and T-cell epitope peptides whichaffect B- and T-cell function in in vitro studies with blood cells fromDer f I or Der f II-sensitive individuals. This can also be applied toDer p I or Der p II, in order to block allergic response to theseallergens.

The cDNA encoding Der f I or Der f II or peptide including at least oneepitope can be used to produce additional peptides, using knowntechniques such as gene cloning. A method of producing a protein or apeptide of the present invention can include, for example, culturing ahost cell containing an expression vector which, in turn, contains DNAencoding all or a portion of a selected allergenic protein or peptide(e.g., Der f I, Der f II or a peptide including at least one epitope).Cells are cultured under conditions appropriate for expression of theDNA insert (production of the encoded protein or peptide). The expressedproduct is then recovered, using known techniques. Alternatively, theallergen or portion thereof can be synthesized using known mechanical orchemical techniques. As used herein, the term protein or peptide refersto proteins of peptides made by any of these techniques. The resultingpeptide can, in turn, be used as described previously.

DNA to be used in any embodiment of this invention can be cDNA obtainedas described herein or, alternatively, can be any oligodeoxynucleotidesequence having all or a portion of the sequence represented in FIGS. 2and 6, or their functional equivalent. Such oligodeoxynucleotidesequences can be produced chemically or mechanically, using knowntechniques. A functional equivalent of an oligonucleotide sequence isone which is capable of hybridizing to a complementary oligonucleotidesequence to which the sequence (or corresponding sequence portions) ofFIGS. 2 and 6 hybridizes and/or which encodes a product (e.g., apolypeptide or peptide) having the same functional characteristics ofthe product encoded by the sequence (or corresponding sequence portion)represented in these figures. Whether a functional equivalent must meetone or both criteria will depend on its use (e.g., if it is to be usedonly as an oligoprobe, it need meet only the first criterion and if itis to be used to produce house dust mite allergen, it need only meet thesecond criterion).

The structural information now available (e.g., DNA, protein/peptidesequences) can also be used to identify or define T cell epitopepeptides and/or B cell epitope peptides which are of importance inallergic reactions to D. farinae allergens and to elucidate themediators or mechanisms (e.g., interleukin-2, interleukin-4, gammainterferon) by which these reactions occur. This knowledge should makeit possible to design peptide-based house dust mite therapeutic agentsor drugs which can be used to modulate these responses.

The present invention will now be further illustrated by the followingExamples, which are not intended to be limiting in any way.

EXAMPLES Example 1

Isolation and Characterization of cDNA Coding for Der f I

MATERIALS AND METHODS

Dermatophagoides farinae culture

Mites were purchased from Commonwealth Serum Laboratories, Parkville,Australia.

Construction of the D. farinae cDNA λgt11 Library

Polyadenylated mRNA was isolated from live D. farinae mites and cDNA wassynthesized by the RNase H method (Gubler, V. and B. J. Hoffman, Gene,25:263-269 (1983)) using a kit (Amersham International, Bucks.). Afterthe addition of EcoRI linkers (New England Biolabs, Beverly, Mass.) thecDNA was ligated to alkaline phosphatase treated λgt11 arms (Promega,Madison, Wis.). The ligated DNA was packaged and plated in E. coli Y1090(r-) to produce a library of 2×10⁴ recombinants.

Isolation of Der f I cDNA Clones from the D. farinae cDNA λgt11 Library

Screening of the library was performed by hybridization with two probescomprising the two Der p I cDNA BamHI fragments 1-348 and 349-857generated by BamHI digestion of a derivative of the Der p I cDNA whichhas had two- BamHI restriction sites inserted between amino acidresidues −1 and 1 and between residues 116 and 117 by site-directedmutagenesis (Chua, K. Y. et al., J. Exp. Med., 167:175-182 (1988)). Theprobes were radiolabelled with ³²P by nick translation. Phage wereplated at 20,000 pfu per 150 mm petri dish and plaques were lifted ontonitrocellulose (Schleicher and Schull, Dassel, F R G), denatured andbaked (Maniatis, T. et al., Molecular cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press (1982)). Prehybridizations were performedfor 2 hours at 42° C. in 50% formamide/5×SSCE/1×Denhardt's/poly C (0.1mg/ml)/poly U(0.1 mg/ml) with hybridization overnight at 42° C. at 10⁶cpm/ml. Post hybridization washes consisted of 15 min washes at roomtemperature with 2×sodium chloride citrate (SSC)/0.1% sodiumdodecylsulphate (SDS), 0.5×SSC/0.1% SDS, 0.1×SSC/0.1% SDS successivelyand a final wash at 50° C. for 30 min in 0.1×SSC/1% SDS.

Isolation of DNA from λyt11 f 1 cDNA Clones

Phage DNA from λgt11 f 1 clones was prepared by a rapid isolationprocedure. Clarified phage plate lysate (1 ml) was mixed with 270 μL of25% wt/vol polyethylene glycol (PEG 6000) in 2.5 M NaCl and incubated atroom temperature for 15 min. The mixture was then spun for 5 min in amicrofuge (Eppendorf, F R G), and the supernatant was removed. Thepellet was dissolved in 100 μL of 10 mM Tris/HCl pH8.0 containing 1 mMEDTA and 100 mM NaCl (TE). This DNA preparation was extracted withphenol/TE, the phenol phase was washed with 100 μl TE, the pooledaqueous phases were then extracted another 2 times with phenol/TE, 2times with Leder phenol (phenol/chloroform/isoamylalcohol; 25:24:1),once with chloroform and the DNA was precipitated by ethanol.

DNA Sequencing

To obtain clones for DNA sequence analysis, the λgt11 f1 phage DNA wasdigested with EcoRI restriction enzyme (Pharmacia, Uppsala, Sweden) andthe DNA insert was ligated to EcoRI-digested M13-derived sequencingvectors mp18 and mp19 (Maniatis, T. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1982)).Transformation was carried out using E. coli TG-1 and sequencing wasperformed by the dideoxynucleotide chain termination method (Sanger, F.et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977)) using theSequenase version 2.0 DNA sequencing kit (U. S. B., Cleveland, Ohio).

Polymerase Chain Reaction(PCR)

PCR was performed by the Taq DNA polymerase method (Saiki, R. K. et al.,Science, 239:487-491 (1988)) using the TaqPaq kit (BiotechInternational, Bentley, Wash.) and the conditions recommended by thesupplier with long of target DNA and 10 pmol of λgt11 primers (NewEngland BioLabs, Beverly, Mass.).

RESULTS

Isolation of Der f I cDNA clones

Two clones expressing the major mite allergen Der f I were isolated fromthe D. farinae cDNA λgt11 library by their ability to hybridize withboth of the Der p I cDNA probes (nucleotides 1-348 and 349-857). Thisapproach was adopted because amino acid sequencing had shown highhomology (80%) between these two allergens (Thomas, W. R. et al.,Advances in the Biosciences, 14:139-147 (1989)). Digestion of the λgt11f1 clone DNA with EcoRI restriction enzyme to release the cDNA insertproduced three Der f I cDNA EcoRI fragments: one approximately 800 baseslong and a doublet approximately 150 bases long. The Der f I cDNA insertwas also amplified from the phage DNA by the polymerase chain reaction(PCR) resulting in a PCR product of approximately 1.1-kb. Each Der f IcDNA fragment was cloned separately into the M13-derived sequencingvectors mp18 and mp19 and sequenced.

DNA Sequence Analysis

The nucleotide sequence of the Der f I cDNA was determined using thesequencing strategy shown in FIG. 1. The complete sequence was shown tobe 1084 bases long and included a 335-base long 5′ proximal endsequence, a coding region for the entire native Der f I protein of 223amino acids with a derived molecular weight of 25,191 and an 80-baselong 3′ noncoding region (FIG. 2). The assignment of the threonineresidue at position 1 as the NH₂-terminal amino acid of Der f I wasbased on data obtained by NH₂-terminal amino acid sequencing of thenative protein and the predicted amino acid sequence of recombinant Derp I (Chua, K. Y. et al., J. Exp. Med., 167:175-182 (1988)). Thepredicted amino acid sequence of the Der f I cDNA in the NH₂-terminalregion matched completely with that determined at the protein level(FIG. 2).

The complete mature protein is coded by a single open reading frameterminating at the TGA stop codon at nucleotide position 1007-1009. Thefirst ATG codon at nucleotide position 42-44 is presumed to be thetranslation initiation site since the subsequent sequence codes for atypical signal peptide sequence.

Amino Acid Sequence Analysis

The amino acid sequence predicted by nucleotide analysis is shown inFIG. 2. As shown in the composite alignment of the amino acid sequenceof mature Der p I and Der f I (FIG. 3), high homology was observedbetween the two proteins. Sequence homology analysis revealed that theDer f I protein showed 81% homology with the Der p I protein aspredicted by previous conventional amino acid sequencing. In particular,the residues making up the active site of Der p I, based on thosedetermined for papain, actinidin, cathepsin H. and cathepsin B, are alsoconserved in the Der f I protein. The residues are glutamine (residue29), glycine, serine and cysteine (residues 33-35), histidine (residue171) and asparagine, serine and tryptophan (residues 191-193) where thenumbering refers to Der f I. The predicted mature Der f I amino acidsequence contains a potential N-glycosylation site (Asn-Thr-Ser) atposition 53-55 which is also present as Asn-Gln-Ser at the equivalentposition in Der p I.

Analysis of the predicted amino acid sequence of the entire Der f I cDNAinsert has shown that, as for other cysteine proteases (FIG. 4), the Derf I protein has pre- and proform intermediates. As previously mentioned,the methionine residue at position −98 is presumed to be the initiationmethionine. This assumption is based on the fact that firstly, the 5′proximal end sequence from residues −98 to −81 is composed predominantlyof hydrophobic amino acid residues (72%), which is the characteristicfeature of signal peptides (Von Heijne, G., EMBO J., 3:2315-2323(1984)). Secondly, the lengths of the presumptive pre- (18 amino acidresidues) and pro-peptides (80 residues) are similar to those for othercysteine proteases (FIG. 4). Most cysteine proteases examined have about120 preproenzyme residues (of which an average of 19 residues form thesignal peptide) with cathepsin B the smallest with 80 (Ishidoh, K. etal., FEBS Letters, 226:32-37 (1987)). Der f I falls within this rangewith a total of 98 preproenzyme residues.

By following the method for predicting signal-sequence cleavage sitesoutlined in Von Beijne, it is proposed that cleavage from the pre-Der fI sequence for proenzyme formation occurs at the signal peptidasecleavage site lying between A1a (−81) and Arg (−80) (Von Heijne, G.,Eur. J. Biochem., 133:17-21 (1988) and J. Mol. Biol., 184:99-105(1985)). Thus, the sequence from residues −98 to −81 codes for theleader peptide while the proenzyme moiety of Der f I begins at residueArg (−80) and ends at residue Glu (−1).

Example 2

Isolation and Characterization of cDNA Coding for Der f II

MATERIALS AND METHODS

Amino acid sequence analysis

Preparation of λgt11 D. farinae cDNA Ligations

D. farinae was purchased from Commonwealth Serum Laboratories,Parkville, Australia, and used to prepare mRNA (polyadenylated RNA) asdescribed (Stewart, G. A. and W. R. Thomas, Int. Arch. Allergy ApplImmunol., 83:384-389 (1987)). The mRNA was suspended at approximately0.5 μg/μl and 5 μg used to prepare cDNA by the RNase H method (Gubler,U. and Hoffman, B. J., Gene, 25:263-269 (1983)) using a kit (AmershamInternational, Bucks). EcoRI linkers (Amersham, GGAATTCC) were attachedaccording to the method described by Huynh et al., Constructing andscreening cDNA libraries in gt10 and gt11, In: Glover, DNA Cloning vol.A practical approach pp. 47-78 IRL Press, Oxford (1985)). The DNA wasthen digested with EcoRI and recovered from an agarose gel purificationby electrophoresis into a DEAE membrane (Schleicher and Schuell, Dassel,FRG, NA-45) according to protocol 6.24 of Sambrook et al., (Sambrook etal., Molecular Cloning, A Laboratory Manual, 2d Ed, Cold Spring HarborLaboratory Press (1989)), except 0.5M arginine base was used forelution. The cDNA was then ligated in λgt10 and λgt11 at an arms toinsert ratio of 2:1. Some was packaged for plaque libraries and analiquot retained for isolating sequences by polymerase chain reaction asdescribed below.

Isolation of Der f II cDNA Polymerase Chain Reaction

To isolate Der f II cDNA, an oligonucleotide primer based on theN-terminal sequence of Der p II was made because their amino acidresidues are identical in these regions (Heymann, P. W. et al., J.Allergy Clin. Immunol., 83:1055-1087 (1989)). The primerGGATCCGATCAACTCGATGC-3′ was used. The first GGATCC encodes a BamH1 siteand the following sequence GAT . . . encodes the first 4 residues of Derp II. For the other primer the λgt11 TTGACACCAGACCAACTGGTAATG-3′ reverseprimer flanking the EcoRI cloning site was used (New England Biolabs,Beverly, Mass.). The Der p II primer was designed to have approximately50-60% G-C and to end on the first or second, rather than the third,base of a codon (Gould, S. J. et al., Proc. Natl. Acad. Sci.,86:1934-1938 (1989); Summer, R. and D. Tautz, Nucleic Acid Res., 17:6749(1989)).

The PCR reactions were carried out in a final reaction volume of 25 μlcontaining 67 mM Tris-HCL (pH8.8 at 25° C.), 16.6 mM (NH₄)₂SO₄, 40 μMdNTPs, 5 mM 2-mercaptoethanol, 6 μM EDTA, 0.2 mg/ml gelatin, 2 mM MgCl₂,10 pmoles of each primer and 2 units of Taq polymerase. Approximately0.001 μg of target DNA was added and the contents of the tube were mixedand overlayed with paraffin oil. The tubes were initially denatured at95° C. for 6 minutes, then annealed at 55° C. for 1 minute and extendedat 72° C. for 2 minutes. Thereafter for 38 cycles, denaturing wascarried out for 30 seconds and annealing and extension as before. In thefinal (40th) cycle, the extension reaction was increased to 10 minutesto ensure that all amplified products were full length. The annealingtemperature was deliberately set slightly lower than the Tm of theoligonucleotide primers (determined by the formula Tm=69.3+0.41(G+C%)−650/oligo length) to allow for mismatches in the N-terminal primer.

5 μl of the reaction was then checked for amplified bands on a 1%agarose gel. The remainder of the reaction mixture was extracted withchloroform to remove all of the paraffin oil and ethanol precipitatedprior to purification of the amplified product on a low melting pointagarose gel (Bio-Rad, Richmond, Calif.).

Subcloning of PCR Product

The ends of the purified PCR product were filled in a reactioncontaining 10 mM Tris HCl, 10 mM MgCl₂, 50 mM NaCl, 0.025 mM dNTP and 1μl of Klenow enzyme in a final volume of 100 μl. The reaction wascarried out at 37° C. for 15 minutes and heat inactivated at 70° C. for10 minutes. The mixture was Leder phenol extracted before ethanolprecipitation. The resulting blunt ended DNA was ligated into M13mp18digested with Sma I in a reaction containing 0.5M ATP, 1×ligase bufferand 1 unit of T₄ ligase at 15° C. for 24 hrs and transformed into E.coli TG1 made competent by the CaCl₂ method. The transformed cells wereplated out as a lawn on L+G plates and grown overnight at 37° C.

Preparation of Single-stranded DNA Template for Sequencing

Isolated white plaques were picked using an orange stick into 2.5 ml ofan overnight culture of TG1 cells diluted 1 in 100 in 2×TY broth, andgrown at 37° C. for 6 hours. The cultures were pelleted and thesupernatant removed to a fresh tube. To a 1 ml aliquot of thissupernatant 270 μl of 20% polyethylene glycol, 2.5 M NaCl was added andthe tube was vortexed before allowing it to stand at RT for 15 minutes.This was then spun down again and all traces of the supernatant wereremoved from the tube. The pellet was then resuspended in 100 μl of 1×TEbuffer. At least 2 phenol:TE extractions were done, followed by 1 Lederphenol extraction and a CHCL₃ extraction. The DNA was precipitated inethanol and resuspended in a final volume of 20 μl of TE buffer.

DNA Analysis

DNA sequencing was performed with the dideoxynucleotide chaintermination (Sanger, F et al., Proc. Nat. Acad. Sci., 74:5463-5467(1977)) using DNA produced from M13 derived vectors mp18 and mp19 in E.coli TG1 and T4 DNA polymerase (Sequenase version 2.0, USB Corp.,Cleveland, Ohio; Restriction endonucleases were from Toyobo, (Osaka,Japan). All general procedures were by standard techniques (Sambrook, J.et al., A Laboratory Manual, 2d Ed. Cold Spring Harbor Laboratory Press(1989)). The sequence analysis was performed using the Mac VectorSoftware (IBI, New Haven, Conn.).

RESULTS

D. farinae cDNA ligated in λgt11 was used to amplify a sequence using anoligonucleotide primer with homology to nucleotides coding for the 4 Nterminal residues of Der p II and a reverse primer for the λgt11sequence flanking the coding site. Two major bands of about 500 bp and300 bp were obtained when the product was gel electrophoresed. Thesewere ligated into M13 mp18 and a number of clones containing the 500 bpfragment were analyzed by DNA sequencing. Three clones produced sequencedata from the N-terminal primer end and one from the other orientation.Where the sequence data from the two directions overlapped, a completematch was found. One of the clones read from the N-terminal primer,contained a one-base deletion which shifted the reading frome. It wasdeduced to be a copying error, as the translated sequence from the othertwo clones matched the protein sequence for the first 20 amino acidresidues of the allergen.

The sequence of the clones showing consensus and producing a correctreading frame is shown in FIG. 6, along with the inferred amino acidsequence. It coded for a 129 residue protein with no N-glycosylationsite and a calculated molecular weight of 14,021 kD. No homology wasfound when compared to other proteins on the GenBank data base (61.0release). It did, however, show 88% amino acid residue homology with Derp II shown in the alignment in FIG. 9. Seven out of the 16 changes wereconservative. The conserved residues also include all the cysteinespresent at positions 8, 21, 27, 73, 78 and 119. There was alsoconsiderable nucleotide homology, although the restriction enzyme mapgenerated from the sequence data for commonly used enzymes is differentfrom Der p II (FIG. 8). The hydrophobicity plots of the translatedsequence of Der f II and Der p II shown in FIG. 10 are almost identical.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. An isolated DNA encoding a peptide from a proteinallergen selected from the group consisting of a Der f I proteinallergen comprising the amino acid sequence shown in FIG. 2 and aDer fII protein allergen comprising the amino acid sequence shown in FIG. 6,wherein the peptide comprises at least one epitope of said proteinallergen, provided that said epitope does not cross-react with a Der p Iprotein allergen or a Der p II protein allergen.
 2. The isolated DNA ofclaim 1 comprising a portion of the nucleotide sequence shown in FIG. 2.3. The isolated DNA of claim 1 comprising a portion of the nucleotidesequence shown in FIG.
 6. 4. The isolated DNA of claim 1 encoding apeptide comprising amino acid residues 34-47 of the amino acid sequenceshown in FIG.
 2. 5. The isolated DNA of claim 1 encoding a peptidecomprising amino acid residues 60-72 of the amino acid sequence shown inFIG.
 2. 6. The isolated DNA of claim 1 encoding a peptide comprisingamino acid residues 166-194 of the amino acid sequence shown in FIG. 2.7. An expression vector comprising the nucleic acid of claim
 1. 8. Theisolated DNA of claim 1, wherein the epitope is a T cell epitope or a Bcell epitope.
 9. A host cell transfected with the expression vector ofclaim
 7. 10. The host cell of claim 9 which is a eukaryotic cell.
 11. Amethod of producing a peptide from a protein allergen selected from thegroup consisting of a Der f I protein allergen and a Der f II proteinallergen, comprising culturing the host cell of claim 10 in medium toexpress the peptide and isolating the peptide from the culture.